Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Patent
1998-06-10
2000-04-25
Prouty, Rebecca E.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
435194, 4353201, 4352523, 436 86, C12N 912, C12Q 148
Patent
active
060542853
DESCRIPTION:
BRIEF SUMMARY
The present invention concerns a method for screening for compounds which are potential modulators of signalling pathways involving the serine/threonine kinase RAC and to the use of the pleckstrin homology domain of RAC-PK in such a method.
The RAC subfamily of serine/threonine protein kinases [Jones, et al. (1991) Proc. Nati. Acad. Sci. U. S. A. 88, 4171-4175; Jones, et al. (1991) Cell Reg. 2, 1001-1009], which are closely related to the cAMP-dependant protein kinase (PKA) and Ca.sup.2+ /phospholipid dependant protein kinase (PKC) families in their kinase domain, contain an amino-terminal pleckstrin homology (PH) domain [Haslam, et al (1993) Nature 363, 309-310]. The human RAC-PK.alpha. isoform is the protooncogenic form of v-akt, which encodes a complete RAC-PK with the addition of a truncated gag sequence at the amino-terminus providing a myristylation sequence.
The PH domain was originally identified as an internal repeat, present at the amino and carboxy-termini of pleckstrin, a 47 kDa protein which is the major PKC substrate in activated platelets [Tyers, et al. (1988) Nature 333, 470-4731]. The superfamily of PH domain containing molecules consists of over 60 members, including serine/threonine protein kinases (RAC-PK, Nrk, .beta.ARK, PKC.mu.), tyrosine protein kinases (Btk, Tec, Itk), GTPase regulators (ras-GAP, ras-GRF, Vav, SOS, BCR), all known mammalian phospholipase Cs, cytoskeletal proteins (.beta.-spectrin, AFAP-110, syntrophin), "adapter" proteins (GRB-7, 3BP2) and kinase substrates (pleckstrin, IRS-1).
While the PH domain structure has been solved for .beta.-spectrin, dynamin and pleckstrins amino-terminal domain, its precise function remains unclear. The presence of PH domains in many signalling and cytoskeletal proteins implicates it in mediating protein-protein interactions. Indeed, the PH domain of the .beta.-adrenergic receptor kinase (.beta.ARK) appears partly responsible for its binding to the .beta..gamma.-subunits of the heterotrimeric G-proteins associated with the .beta.-adrenergic receptor, while the PH domain of the Bruton's tyrosine kinase (Btk) appears to mediate an interaction with PKC. Several PH domains have been shown to be able to bind phosphatidyl-inositol-(4,5)-bisphosphate in vitro, although weakly. We have now shown that the PH domain of RAC-PK binds phospholipid with high affinity, which suggests that RAC-PK my be membrane-bound in vivo via the PH domain. Moreover, binding of phospholipid to the RAC-PK PH domain quenches the intrinsic Trp fluorescence thereof.
SUMMARY OF THE INVENTION
According to a first aspect of the invention, therefore, we provide a method for screening a compound which is a candidate modulator of signal response comprising the steps of: which is capable of fluorescing; the PH domain, an alteration of the fluorescence in the presence of the compound being indicative of a functional interaction between the compound and the PH domain.
DETAILED DESCRIPTION OF THE INVENTION
The observed high affinity binding of phospholipid to the N-terminus of the PH domain of signalling molecules suggests that the PH domain functions as a membrane anchor for the molecules. Interaction of molecules with the cell membrane is believed to be important for the stable interaction thereof with membrane bound partners in signalling pathways, such that disruption of this interaction will lead to modulation of the signalling effect through the dissociation of the signalling molecule from the cell membrane. The modulation could be either down-regulating, for example if the otherwise stable interaction of the molecule with membrane-bound partners is a stimulatory interaction, or up-regulating, in the event that the interaction is an inhibitory interaction.
Accordingly, compounds which affect the binding of the PH domain to phospholipids present in the cell membrane are potential modulators of signal response.
Such compounds could be agents which cause a conformational change in the PH domain, either increasing or decreasing its affinity for phospholipid, or compounds wh
REFERENCES:
patent: 5266565 (1993-11-01), Lacoste et al.
patent: 5278154 (1994-01-01), Lacoste et al.
patent: 5385915 (1995-01-01), Buxbaum et al.
patent: 5422125 (1995-06-01), Skyler et al.
Boudewijn Burgering et al., Laboratory for Physiological Chemistry, Nature--vol. 376, pp. 599-603.
Kohn et al., The EMBO Journal, vol. 14, No. 17, pp. 4288-4295, 1995.
Jeno et al., Proc. Natl. Acad. Sci. USA, vol. 85, pp. 406-410, Jan. 1988.
Chambers et al., Molecular Pharmacology, vol. 41, No. 6, pp. 1008-1015, 1992.
Persaud et al., Biochem J., vol. 319, pp. 119-124, (1996).
Carey et al., Diabetes, vol. 44, 1995, pp. 682-688, Jun. 1995.
Andjelkovic et al., Proc. Natl. Acad. Sci. USA, vol. 93, pp. 5699-5704, Jun. 1996.
Derwent Abstract 91-019158/03, JP 02292217, Aug. 5, 1989.
Jones et al., Cell Regulation, vol. 2, pp. 1001-1009, Dec. 1991.
Tyers et al., Nature vol. 333, pp. 470-473, Jun. 1988.
Cross et al., Nature, vol. 378, vol. 21/28, pp. 785-789 (1995).
Franke et al., Cell, vol. 81, pp. 727-736 (1995).
Frech Matthias
Hemmings Brian Arthur
Ferraro Gregory D.
Monshipouri M.
Novartis AG
Prouty Rebecca E.
LandOfFree
Screening method does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Screening method, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Screening method will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-992370